1. Field of the Invention
The present invention relates to a method for enriching nucleic acids with a length of less than 300 nucleotides, to a kit for enriching nucleic acids with a length of less than 300 nucleotides, to the use of said kit, to the use of an anion exchange matrix and to a method for treating a disease.
2. Description of Related Art
Since small ribonucleic acids (RNAs) were found, a few years ago, to carry out an essential regulatory function in gene expression, scientific studies have increasingly concentrated on small RNAs shorter than 300 nucleotides, in particular shorter than 100 nucleotides. More specifically, many researchers have become interested in micro RNAs (miRNAs). miRNAs are an evolutionarily conserved class of small noncoding RNAs of about 22 nucleotides in length, whose complex role during regulation of gene expression is becoming more and more obvious. miRNAs have been found in any eukaryotic organism studied and in nearly every tissue, inter alia in fungi, plants, insects and mammals. Aside from miRNAs, other small RNAs have also been found which likewise play a fundamental role in cellular functions. These include, for example, small interfering RNAs (siRNAs), small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs).
These short RNAs which are shorter than 300 nucleotides must be as pure as possible and purified with high yield from the biological systems to be studied in order for it to be possible for their cellular role to be investigated. There is therefore a great need to provide a method which enables such short RNAs to be purified or isolated from complex biological systems, in particular from cell lysates.
Generally, in order to isolate nucleic acids from biological samples, they must be separated from the remaining cellular components such as proteins, sugars, lipids and other components. The prior art has disclosed a plurality of methods of separating nucleic acids from very different starting materials, for example from cell cultures, from tissues of plant and animal origin and from body fluids. One method, for example, includes extracting the usually aqueous starting solutions with the aid of organic solvents such as phenol and chloroform (Chomczynski and Sacchi, 1987) followed by precipitation of the nucleic acids with the aid of alcohols such as ethanol or isopropanol from the aqueous phase (Sambrook, J., Fritsch, E. F. in T. Maniatis, CSH, “Molecular Cloning”, 1989). Another method comprises immobilizing the nucleic acids to a solid phase, for example by means of silica adsorption technology. Disadvantageously, it is not, or only insufficiently, possible in all of these methods to isolate or at least purify relatively small nucleic acids.
To solve this problem, the prior art has disclosed methods for specifically enriching small RNA populations, which methods are based on silica membrane technology. In these methods, a specific, relatively small amount of alcohol is added to the cell lysate after the cells have been lysed, and at least part of the relatively long nucleic acids are bound to the silica membrane under chaotropic binding conditions. However, the amount of alcohol in the purification methods described in the prior art is too low to bind efficiently also small nucleic acids to the silica membrane, and these small RNAs are therefore present in the breakthrough. The alcohol concentration is then increased in the breakthrough and the latter is bound to a second silica membrane. After washing steps, the small RNA is eluted together with all other nucleic acids that have not been bound to the first column (see, for example, mirVana® kit from Ambion, Austin, USA or RNeasy® Lipid Tissue Mini Kit from QIAGEN, Hilden, Germany, to be used by means of the “user developed protocols”).
The disadvantage of both the QIAGEN and the Ambion methods is that of two solid phases having to be used and it not being possible to obtain only the desired small RNA with the aid of a single binding step. Moreover, this method does not enable, for example, only miRNAs of about 22 nucleotides in size to be isolated, without isolating also tRNAs and other larger nucleic acids at the same time. Although it is possible to enrich the small RNA, in particular the miRNA, to a certain degree by this method under some circumstances, said small RNA is still contaminated with other nucleic acids, in particular with transfer RNA (tRNA).